Receptor-Mediated Activation of Heterotrimeric G-Proteins in Living Cells

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Science  23 Mar 2001:
Vol. 291, Issue 5512, pp. 2408-2411
DOI: 10.1126/science.1055835

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Receptor-mediated activation of heterotrimeric GTP–binding proteins (G-proteins) was visualized in living Dictyostelium discoideum cells by monitoring fluorescence resonance energy transfer (FRET) between α- and β- subunits fused to cyan and yellow fluorescent proteins. The G-protein heterotrimer rapidly dissociated and reassociated upon addition and removal of chemoattractant. During continuous stimulation, G-protein activation reached a dose-dependent steady-state level. Even though physiological responses subsided, the activation did not decline. Thus, adaptation occurs at another point in the signaling pathway, and occupied receptors, whether or not they are phosphorylated, catalyze the G-protein cycle. Construction of similar energy-transfer pairs of mammalian G-proteins should enable direct in situ mechanistic studies and applications such as drug screening and identifying ligands of newly found G-protein–coupled receptors.

  • * Present address: Department of Cell Biology and Anatomy, Johns Hopkins Medical Institutions, Baltimore, MD 21205, USA.

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